Hydration changes accompanying the binding of minor groove ligands with DNA - PubMed (original) (raw)
Hydration changes accompanying the binding of minor groove ligands with DNA
Natalya N Degtyareva et al. Biophys J. 2007.
Abstract
4',6-diamidino-2-phenylindole (DAPI), netropsin, and pentamidine are minor groove binders that have terminal -C(NH2)2+ groups. The hydration changes that accompany their binding to the minor groove of the (AATT)2 sequence have been studied using the osmotic stress technique with fluorescence spectroscopy. The affinity of DAPI for the binding site decreases with the increasing osmolality of the solution, resulting in acquisition of 35+/-1 waters upon binding. A competition fluorescence assay was utilized to measure the binding constants and hydration changes of the other two ligands, using the DNA-DAPI complex as the fluorescence reporter. Upon their association to the (AATT)2 binding site, netropsin and pentamidine acquire 26+/-3 and 34+/-2 additional waters of hydration, respectively. The hydration changes are discussed in the context of the terminal functional groups of the ligands and conformational changes in the DNA.
Figures
FIGURE 1
Molecular structures of (a) DAPI, (b) netropsin, and (c) pentamidine.
FIGURE 2
(A) Binding isotherms for the titration of the DNA into 10 nM DAPI in buffers with 0.5 M (crosses), 1.5 m (open circles), and 2.5 m (open squares) triethylene glycol show that the affinity of DAPI for the (AATT)2 binding site decreases with increasing concentrations of triethylene glycol. (B) Plot of the natural logarithm of the observed equilibrium constant for the association of DAPI with the DNA as a function of the concentrations of acetamide (open circles), betaine (open squares), triethylene glycol (solid circles), and trimethylamine N-oxide (solid squares). A linear least-squares fit using Eq. 5 gives 35 ± 1 waters that are acquired by the complex. The extrapolation to the buffer conditions gives 6.7 (± 0.1) × 108 M−1.
FIGURE 3
(A) Binding isotherm for the titration of netropsin to the DAPI-DNA complex in a buffer with 1.1 m triethylene glycol. The solid line is the fit using Eq. 4 which yields a relative equilibrium constant of 7.6 ± 0.6. The dashed line is the emission intensity of the unbound DAPI before the addition of the DNA. (B) Plot of the natural logarithm of the relative equilibrium constants for the exchange reactions (Eq. 2) with netropsin as a function of the osmolality of acetamide (open circles), betaine (open squares), triethylene glycol (solid circles), and trimethylamine N-oxide (solid squares). The change in the amount of water is −9 ± 3 for netropsin. The extrapolation to the buffer conditions gives a relative equilibrium constant of 5.0 ± 0.3, which is consistent with the direct measurement of 4.9 ± 1.1 in the buffer alone.
FIGURE 4
(A) Binding isotherm for the titration of pentamidine to the DAPI-DNA complex in a buffer with 1.2 m triethylene glycol. The solid line is the fit using Eq. 4 which yields a relative equilibrium constant of 0.0023 ± 0.0002. The dashed line is the emission intensity of the unbound DAPI before the addition of DNA. (B) Plot of the natural logarithm of the equilibrium constants for the exchange reactions (Eq. 2) with pentamidine as a function of the osmolality of acetamide (open circles), betaine (open squares), triethylene glycol (solid circles), and trimethylamine N-oxide (solid squares). The change in the amount of water is −1 ± 2 for pentamidine. The extrapolation to the buffer conditions gives a relative equilibrium constant of 2.0 (± 0.5) × 10−3, which is consistent with the direct measurement of 1.9 (±0.2) × 10−3 in buffer alone.
FIGURE 5
Integrated heats from the calorimetric titration of 500 _μ_M pentamidine into BPES-50 (circles) and 6 _μ_M (CGCGCAATTGCGCG)2 (triangles) in BPES-50 at 25°C. The one-site model yielded a heat of reaction of −2.4 ± 0.2 kcal/mol from the average of three separate titrations. The heat of dilution is −1.9 ± 0.1 kcal/mol. The heat of reaction for the addition to the DNA solution approaches the heat of dilution after saturation of the minor groove binding site. The titration to the DNA was continued to 11 pentamidines:oligonucleotide with no change in the heat of reaction.
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